The goal of this project is to investigate the pathogenic mechanisms underlying the neurodegeneration of amyotrophic lateral sclerosis (ALS) using transgenic mouse models. Our application has two immediate Aims: The first one is to replace the two free cysteine residues in mutant human SOD 1 protein to test the role of free -SH groups in the formation of intracellular aggregates noted in ALS. The second aim is to define the smallest fragment of SOD 1 that would still cause ALS in transgenic mice. AIM 1: About 20 percent of the familial ALS cases are caused by mutations in Cu/Zn superoxide dismutase gene (SOD 1). Transgenic mice that over express mutant SOD 1 develop an ALS-like phenotype and motor neuron degeneration. A common feature in the pathology of both human SOD1-linked ALS and the ALS (SOD1) mouse models is the presence of the SOD 1-inimunoreactive inclusions or aggregates in neurons of the brain and spinal cord. These inclusions/aggregates are thought to be important elements in motor neuron death in ALS. To test the hypothesis that these inclusions/aggregates may be formed by disulfide bonds through interaction of free -SH groups of one or both free cysteines in SOD 1, we propose to develop new transgenic mouse model that over expresses a mutated SOD 1 (C6A/C1 1 1S/G93A). In this transgenic mouse model, two free cysteines in human SOD1 are replaced by an alanine and a serine. Absence of inclusions/aggregates will indicate a role for free cysteines; Absence of disease as well as inclusions/aggregates may indicate a causative role of the free cysteines (-SH groups). AIM 2: We recently made a new transgenic mouse model that over expresses a truncation mutation in SOD 1 (L126Z). These mice developed a typical ALS-like phenotype and pathology. These results provide the experimental evidence that only a part of the SOD1 polypeptide, rather than entire SOD1 protein, is sufficient to produce the neuronal toxicity and cause motor neuron degeneration. We plan to define the minimum fragment of SOD 1 essential for toxicity so that further studies into the pathogenesis of ALS may be facilitated. To achieve this goal we propose to develop additional transgenic mouse lines that over express successively smaller fragments of SOD 1 polypeptide to determine the smallest segment of SOD 1 that causes ALS.